This revised R21 exploratory proposal focuses on the interaction of alcohol with hepatitis C virus (HCV) proteins in the pathogenesis of HCV liver disease. HCV infection is a significant clinical problem and is a major cause of liver disease worldwide. The prevalence of HCV infection is significantly higher in alcohol abusers, who are also at greatly increased risk for development of liver diseases that include fibrosis, cirrhosis and hepatocellular carcinoma. Moreover, the incubation period for development of liver disease is much shorter in HCV-infected alcohol abusers. HCV infected patients who abuse alcohol also have reduced responses to interferon (IFN) therapy. Alcohol and HCV proteins have separately been shown to induce a plethora of changes in signal transduction cascades in human cells, including inhibition of the IFN system and increased expression of pro-inflammatory cytokines and chemokines, such as TNF-alpha and IL-8. Our preliminary data indicate that the HCV NS5A protein induces IL-8 to inhibit the IFN system, while the HCV core protein activates the IFN system. However, very little is known about how alcohol affects cellular signal transduction pathways in the context of HCV protein expression, and the resultant effects on liver cell horneostasis. We hypothesize that alcohol and HCV proteins induce pro-inflammatory cytokine and chemokine expression, inhibit IFN signal transduction, and perturb the expression of other cellular proteins and signal transduction pathways in human hepatocytes. Using human hepatocyte cell lines stably expressing the HCV NS5A and core proteins under tetracycline regulation, this exploratory application will address two specific aims. Specific Aim 1 will determine the effects of physiological concentrations of ethanol with HCV core and NS5A protein expression on pro-inflammatory cytokine and chemokine expression, and IFN signal transduction. Specific Aim 2 will characterize the global transcriptional changes in human hepatocytes induced by HCV core and NS5A proteins, ethanol, and IFN by CDNA microarray analysis. These studies will provide mechanistic information on how alcohol interacts with the HCV-infected patient to promote liver disease, and may lead to strategies to counteract the deleterious effects of these agents on the liver.